Plants and Seeds of Hybrid Corn Variety CH011213

ABSTRACT

According to the invention, there is provided seed and plants of the hybrid corn variety designated CH011213. The invention thus relates to the plants, seeds and tissue cultures of the variety CH011213, and to methods for producing a corn plant produced by crossing a corn plant of variety CH011213 with itself or with another corn plant, such as a plant of another variety. The invention further relates to genetic complements of plants of variety CH011213.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to corn seed and plants of thehybrid variety designated CH011213, and derivatives and tissue culturesthereof.

Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Plant breeding techniques take advantage of how a plant is naturallypollinated. There are two general methods of pollination. A plant isself-pollinated when pollen from one flower is transferred to the sameflower or another flower of the same plant. A plant is cross-pollinatedwhen pollen comes to it from a flower of a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,which are located on the tassel and the ear, respectively. Naturalpollination occurs in corn when the wind blows pollen from the tasselsto the silks that protrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, i.e., a homozygous plant. Across between two such homozygous plants produces a uniform populationof hybrid plants that are heterozygous for many gene loci andphenotypically uniform.

The development of uniform corn plant hybrids requires developinghomozygous inbred plants, crossing these inbred plants, and evaluatingthese crosses. Pedigree breeding and recurrent selection are examples ofbreeding methods used to develop hybrid parent plants from breedingpopulations. Those breeding methods combine the genetic backgrounds fromtwo or more inbred plants or various other broad-based sources intobreeding pools from which new inbred plants are developed by selfingcombined with phenotypic or genotypic selection. The new inbred plantsare crossed with other inbred plants and the hybrids produced by thesecrosses are evaluated for commercial potential.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of the hybridvariety designated CH011213. Also provided are corn plants having allthe physiological and morphological characteristics of the hybrid cornvariety CH011213. A hybrid corn plant of the invention may furthercomprise a cytoplasmic or nuclear factor that is capable of conferringmale sterility or otherwise preventing self-pollination, such as byself-incompatibility. Parts of the corn plant of the present inventionare also provided, for example, pollen obtained from a hybrid plant andan ovule of the hybrid plant. The invention also concerns seed of thehybrid corn variety CH011213. The hybrid corn seed of the invention maybe provided as a population of corn seed of the variety designatedCH011213.

In a further aspect, the invention provides a composition comprising aseed of corn variety CH011213 comprised in plant seed growth media. Incertain embodiments, the plant seed growth media is a soil or syntheticcultivation medium. In specific embodiments, the growth medium may becomprised in a container or may, for example, be soil in a field.

In another aspect of the invention, the hybrid corn variety CH011213comprising an added heritable trait or genetic modification is provided.The heritable trait may comprise a genetic locus that comprises adominant or recessive allele. In certain embodiments of the invention,the genetic locus confers traits such as, for example, male sterility,waxy starch, herbicide resistance, insect resistance, resistance tobacterial, fungal, nematode or viral disease, and altered fatty acid,phytate or carbohydrate metabolism. In certain embodiments, the geneticlocus that confers herbicide resistance may confer resistance toherbicides such as, for example, imidazolinone herbicides, sulfonylureaherbicides, triazine herbicides, phenoxy herbicides, cyclohexanedioneherbicides, benzonitrile herbicides, 4-hydroxyphenylpyruvatedioxygenase-inhibiting herbicides, protoporphyrinogen oxidase-inhibitingherbicides, acetolactate synthase-inhibiting herbicides,1-aminocyclopropane-1-carboxylic acid synthase-inhibiting herbicides,bromoxynil, nicosulfuron, 2,4-dichlorophenoxyacetic acid (2,4-D),dicamba, quizalofop-p-ethyl, glyphosate, or glufosinate. The geneticlocus may be a naturally occurring corn gene introduced into the genomeof a parent of the variety by backcrossing, a natural or inducedmutation, or a transgene introduced through genetic transformationtechniques. When introduced through transformation, a genetic locus maycomprise one or more transgenes integrated at a single chromosomallocation.

In further embodiments, a single locus conversion of one or both of theparental varieties of the hybrid corn variety CH011213 comprises aheritable genetic modification to the genome of one or both of theparental varieties. A heritable genetic modification may comprise, forexample, an insertion, deletion, or substitution of a nucleotidesequence. In certain embodiments, a single locus may comprise one ormore genes or intergenic regions integrated into or mutated at a singlelocus or may comprise one or more nucleic acid molecules integrated atthe single locus. In particular embodiments, a single locus conversionmay be generated by genome editing such as through use of engineerednucleases, as is known in the art. Examples of engineered nucleasesinclude, but are not limited to, Cas endonucleases, zinc fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), and engineered meganucleases, also known as homingendonucleases. Naturally occurring nucleases can also find use forgenome editing. In specific embodiments, endonucleases, both naturallyoccurring and engineered, may utilize any polypeptide-, DNA-, orRNA-guided genome editing systems known to the skilled artisan.

In yet another aspect of the invention, a hybrid corn plant of thevariety designated CH011213 is provided, wherein acytoplasmically-inherited trait has been introduced into said hybridplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continues to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety CH011213 is provided. The tissue culturewill preferably be capable of regenerating plants capable of expressingall of the physiological and morphological characteristics of thevariety, and of regenerating plants having substantially the samegenotype as other plants of the variety. Examples of some of thephysiological and morphological characteristics of the variety CH011213include characteristics related to yield, maturity, and kernel quality,each of which is specifically disclosed herein. The regenerable cells insuch tissue cultures may, for example, be derived from embryos,meristematic cells, immature tassels, microspores, pollen, leaves,anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, orstalks, or from callus or protoplasts derived from those tissues. Stillfurther, the present invention provides corn plants regenerated from thetissue cultures of the invention, the plants having all thephysiological and morphological characteristics of variety CH011213.

In still another aspect, the invention provides a method of producinghybrid corn seed comprising crossing a plant of variety CV621679 with aplant of variety CV512858. In a cross, either parent may serve as themale or female. Processes are also provided for producing corn seeds orplants in which the processes generally comprise crossing a first parentcorn plant with a second parent corn plant, wherein at least one of thefirst or second parent corn plants is a plant of the variety designatedCH011213. In such crossing, either parent may serve as the male orfemale parent. These processes may be further exemplified as processesfor preparing hybrid corn seed or plants, wherein a first hybrid cornplant is crossed with a second corn plant of a different, distinctvariety to provide a hybrid that has, as one of its parents, the hybridcorn plant variety CH011213. In these processes, crossing will result inthe production of seed. The seed production occurs regardless of whetherthe seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting, often in pollinating proximity, seeds of a first andsecond parent corn plant, and in many cases, seeds of a first corn plantand a second, distinct corn plant. When the plants are not inpollinating proximity, pollination can nevertheless be accomplished bytransferring a pollen or tassel bag from one plant to the other asdescribed below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers (corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant). A third step comprisespreventing self-pollination of the plants, i.e., preventing the silks ofa plant from being fertilized by any plant of the same variety,including the same plant. This can be done, for example, by emasculatingthe male flowers of the first or second parent corn plant, (i.e.,treating or manipulating the flowers so as to prevent pollen production,in order to produce an emasculated parent corn plant).Self-incompatibility systems may also be used in some hybrid crops forthe same purpose. Self-incompatible plants still shed viable pollen andcan pollinate plants of other varieties but are incapable of pollinatingthemselves or other plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant is nolonger viable, and that the only pollen transferred comes from the firstplant. The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated CH011213. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation hybrid corn seed and plants produced bycrossing an inbred with another, distinct inbred. The present inventionfurther contemplates seed of an F₁ hybrid corn plant. Therefore, certainexemplary embodiments of the invention provide an F₁ hybrid corn plantand seed thereof, specifically the hybrid variety designated CH011213.

Such a plant can be analyzed by its “genetic complement.” This term isused to refer to the aggregate of nucleotide sequences, the expressionof which defines the phenotype of, for example, a corn plant, or a cellor tissue of that plant. A genetic complement thus represents thegenetic makeup of a cell, tissue or plant. The invention thus providescorn plant cells that have a genetic complement in accordance with thecorn plant cells disclosed herein, and plants, seeds and diploid plantscontaining such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., marker typing profiles.It is known in the art that such complements may also be identified bymarker types including, but not limited to, Simple Sequence Repeats(SSRs), Simple Sequence Length Polymorphisms (SSLPs) (Williams et al.,Nucleic Acids Res., 18:6531, 1990), Randomly Amplified Polymorphic DNAs(RAPDs), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction(AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs) (EuropeanPatent Application Publication No. EP0534858), and Single NucleotidePolymorphisms (SNPs) (Wang et al., Science, 280:1077, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a corn plant of the invention with a haploid geneticcomplement of the same or a different variety. In another aspect, thepresent invention provides a corn plant regenerated from a tissueculture that comprises a hybrid genetic complement of this invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions of PlantCharacteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. The rating multiplied by 10 isapproximately equal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating. A numerical rating that is basedon a 1 to 9 scale of severity in which “1” indicates “most resistant”and “9” indicates “most susceptible.”

Cn: Corynebacterium nebraskense rating. The rating multiplied by 10 isapproximately equal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. The rating multiplied by 10 isapproximately equal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating. A rating in which the valueequals percent plants girdled and stalk lodged.

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root rating. A rating that is based on a 1 to 9scale in which “1” indicates “least affected” and “9” indicates “severepruning.”

Ear-Attitude: The attitude or position of the ear at harvest, which isscored as upright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, which is scored as white, pink,red, or brown.

Ear-Cob Diameter: The average diameter of the cob when measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, which is scored as strong or weak.

Ear-Diameter: The average diameter of the ear when measured at themidpoint.

Ear-Dry Husk Color: The color of the husks at harvest, which is scoredas buff, red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination, which is scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf, which is scored asshort, medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks in which the minimum value is no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest, which isscored as tight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence, which is scored as green-yellow, yellow, pink, red, orpurple.

Ear-Taper (Shape): The taper or shape of the ear, which is scored asconical, semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating. A rating in which the value approximates percent earrotted.

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units. GDUs are calculated by the Barger Method inwhich the heat units for a 24 h period are calculated as follows:[(Maximum daily temperature+Minimum daily temperature)/2]−50. Thehighest maximum daily temperature used is 86° F. and the lowest minimumtemperature used is 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for a variety to have approximately 50% of the plants sheddingpollen as measured from time of planting. GDUs to shed is determined bysumming the individual GDU daily values from the planting date to thedate of 50% pollen shed.

GDUs to Silk: The number of growing degree units (GDUs) for a variety tohave approximately 50% of the plants with silk emergence as measuredfrom the time of planting. GDUs to silk is determined by summing theindividual GDU daily values from the planting date to the date of 50%silking.

Hc2: Helminthosporium carbonum race 2 rating. The rating multiplied by10 is approximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. The rating multiplied by10 is approximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. The rating multiplied by 10is approximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. The rating multiplied by10 is approximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. The rating multiplied by10 is approximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. “+” indicates the presence of Htchlorotic-lesion type resistance; “−” indicates absence of Htchlorotic-lesion type resistance; and “+/−” indicates segregation of Htchlorotic-lesion type resistance. The rating multiplied by 10 isapproximately equal to percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone, which is scored aswhite, pink, tan, brown, bronze, red, purple, pale purple, colorless, orvariegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,which is scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm, which is scored aswhite, pale yellow, or yellow.

Kernel-Endosperm Type: The type of endosperm, which is scored as normal,waxy, or opaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp, which is scored ascolorless, red-white crown, tan, bronze, brown, light red, cherry red,or variegated.

Kernel-Row Direction: The direction of the kernel rows on the ear, whichis scored as straight, slightly curved, spiral, or indistinct(scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, which is scored as white, pale yellow, yellow, orange, red, orbrown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel, which is scored as dent, flint, orintermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. The rating multiplied by 10 is approximatelyequal to percent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk, which is scored asupright (0 to 30 degrees), intermediate (30 to 60 degrees), or lax (60to 90 degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollination,which is scored as light green, medium green, dark green, or very darkgreen.

Leaf-Length: The average length of the primary ear leaf

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination, which is rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, which is scored as absent,basal-weak, basal-strong, weak, or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf when measured atits widest point.

LSS: Late season standability. The value multiplied by 10 isapproximately equal to percent plants lodged in disease evaluationplots.

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating. The rating is based on a 1 to9 scale in which “1” indicates “resistant” and “9” indicates“susceptible.”

On2: Ostrinia nubilalis 2nd brood rating. The rating is based on a 1 to9 scale in which “1” indicates “resistant” and “9” indicates“susceptible.”

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale in which the best and worst ratings are “1” and “9”,respectively. The score is taken when the average entry in a trial is atthe fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating. The rating is actual percentinfection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear when measured from theground to the point of attachment of the ear shank of the top developedear to the stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant when measured fromthe soil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity) and is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis in which “1” and “9”are the best and worst score, respectively.

STR: Stalk rot rating. The rating is based on a 1 to 9 scale of severityin which “1” indicates “25% of inoculated internode rotted” and “9”indicates “entire stalk rotted and collapsed.”

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating. The numerical rating isbased on a 1 to 9 scale of severity in which “1” indicates “mostresistant” and “9” indicates “most susceptible.”

Tassel-Anther Color: The color of the anthers at 50% pollen shed, whichis scored as green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination, which isscored as open or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel, which is scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glume,which is scored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed, which is scoredas green, red, or purple.

Tassel-Length: The length of the tassel, which is measured from the baseof the bottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle, whichis measured from the base of the flag leaf to the base of the bottomtassel branch.

Tassel-Pollen Shed: A visual rating of pollen shed that is determined bytapping the tassel and observing the pollen flow of approximately fiveplants per entry. The rating is based on a 1 to 9 scale in which “9”indicates “sterile” and “1” indicates “most pollen.”

Tassel-Spike Length: The length of the spike, which is measured from thebase of the top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genomic Selection (GS) or Genome-wide selection (GWS): a use ofgenome-wide genotypic data to predict genomic estimated breeding values(GEBV) for selection purposes in breeding process.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Marker: A readily detectable phenotype or genotype, preferably inheritedin codominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Marker assisted breeding or marker assisted selection (MAS): A processof selecting a desired trait or desired traits in a plant or plants bydetecting one or more markers from the plant, where the marker isassociated with the desired trait.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, to certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing or by genome editing of alocus, wherein essentially all of the morphological and physiologicalcharacteristics of an inbred are recovered in addition to thecharacteristics conferred by the single locus transferred into theinbred via the backcrossing or genome editing technique. A single locusmay comprise one gene, or in the case of transgenic plants, one or moretransgenes integrated into the host genome at a single site (locus).

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Three-way cross hybrid: A hybrid plant produced by crossing a firstinbred plant with the F₁ hybrid progeny derived from crossing a secondinbred plant with a third inbred plant.

Transgene: A genetic sequence which has been introduced into the nuclearor cytoplasmic components of the genome of a corn plant by a genetictransformation technique.

Variety Descriptions

In accordance with one aspect of the present invention, there isprovided a novel hybrid corn plant variety designated CH011213. Hybridvariety CH011213 was produced from a cross of the inbred varietiesdesignated CV621679 and CV512858. The inbred parents have beenself-pollinated and ear-rowed a sufficient number of generations withcareful attention paid to uniformity of plant type to show uniformityand stability within the limits of environmental influence.

In accordance with one aspect of the invention, there is provided a cornplant having the physiological and morphological characteristics of cornplant CH011213. An analysis of such morphological traits was carriedout, the results of which are presented in Table 1.

TABLE 1 Morphological Traits for Hybrid Variety CH011213 CHARACTERISTICVALUE 1 STALK Plant Height (cm) 299   Ear Height (cm) 120.5  AnthocyaninAbsent Brace Root Color Absent Internode Direction Zig - Zag InternodeLength (cm) 18.3 2 LEAF Color Dark Green Length (cm) 93.8 Width (cm) 9.4 Sheath Anthocyanin Absent Sheath Pubescence Medium Marginal WavesModerate Longitudinal Creases Few 3 TASSEL Length (cm) 49.3 PeduncleLength (cm) 14   Branch Number 5  Anther Color Salmon Glume Color Green& Medium Green Glume Band Absent 4 EAR Silk Color Tan Number Per Stalk1  Position Pendent Length (cm) 17.3 Shape Cylindrical Diameter (cm) 4.9 Shank Length (cm)  5.2 Husk Bract Short Husk Cover (cm) 2  HuskOpening Tight Husk Color Fresh Green & Medium Green Husk Color Dry BuffCob Diameter (cm)  2.6 Cob Color Red Shelling Percent 91.7 5 KERNEL RowNumber 16   Number Per Row 35.4 Row Direction Straight Type Dent CapColor Yellow - Orange Side Color Yellow Length (depth) (mm) 13.3 Width(mm)  8.7 Thickness (mm)  4.5 Endosperm Type Normal Endosperm ColorYellow *These are typical values. Values may vary due to environment.Other values that are substantially equivalent are within the scope ofthe invention. Substantially equivalent refers to quantitative traitsthat when compared do not show statistical differences of their means.

In accordance with another aspect of the present invention, there isprovided a corn plant having the morphological characteristics of cornplant CV512858. A description of the morphological and physiologicalcharacteristics of corn plant CV512858 is presented in Table 2.

TABLE 2 Morphological and Physiological Traits for Corn Variety CV512858CHARACTERISTIC VALUE 1 STALK Plant Height (cm) 200.1  Ear Height (cm)73.8 Anthocyanin Basal Weak Brace Root Color Absent Internode DirectionStraight Internode Length (cm) 12.7 2 LEAF Color Green & Medium GreenLength (cm) 85.9 Width (cm) 10.1 Sheath Anthocyanin Absent SheathPubescence Absent Marginal Waves Few Longitudinal Creases Few 3 TASSELLength (cm) 38.2 Peduncle Length (cm)  7.3 Branch Number  7.9 AntherColor Yellow Glume Color Green & Medium Green Glume Band Absent 4 EARSilk Color Yellow Number Per Stalk 2  Position Upright Length (cm) 15.1Shape Cylindrical Diameter (cm)  4.6 Shank Length (cm)  6.7 Husk BractShort Husk Cover (cm)  3.4 Husk Opening Tight Husk Color Fresh Green &Medium Green Husk Color Dry Buff Cob Diameter (cm) 3  Cob Color RedShelling Percent 86.9 5 KERNEL Row Number 16.3 Number Per Row 27.2 RowDirection Straight Type Dent Cap Color Yellow Side Color Deep YellowLength (depth) (mm) 11.5 Width (mm)  8.4 Thickness (mm)  5.2 EndospermType Normal Endosperm Color Yellow *These are typical values. Values mayvary due to environment. Other values that are substantially equivalentare within the scope of the invention. Substantially equivalent refersto quantitative traits that when compared do not show statisticaldifferences of their means.

Deposit Information

A deposit of at least 625 seeds of inbred parent plant varietiesCV621679 (U.S. Pat. 10,448,593) and CV512858 (U.S. patent applicationSer. No 17/517,379, filed Nov. 2, 2021) has been made with either theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209 USA, or the Provasoli-Guillard National Centerfor Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for OceanSciences, 60 Bigelow Drive, East Boothbay, Me. 04544 USA, and assignedATCC Accession No. PTA-125246 and NCMA Accession No. 202106028,respectively. The dates of deposit with the specific InternationalDepositary Authority are Sep. 20, 2018 and Jun. 8, 2021, respectively.All restrictions upon the deposits have been removed, and the depositsare intended to meet all of the requirements of the Budapest Treaty and37 C.F.R. §1.801-1.809. Access to the deposits will be available duringthe pendency of the application to the Commissioner of Patents andTrademarks and persons determined by the Commissioner to be entitledthereto upon request. The deposits have been accepted under the BudapestTreaty and will be maintained in the specific Depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Applicant does not waive any infringement of their rights granted underthis patent or under the Plant Variety Protection Act (7 U.S.C. 2321 etseq.).

Further Embodiments of the Invention

In one embodiment, compositions are provided comprising a seed of cornvariety CH011213 comprised in plant seed cultivation media. Plant seedcultivation media are well known to those of skill in the art andinclude, but are in no way limited to, soil or synthetic cultivationmedium. Plant seed cultivation media can provide adequate physicalsupport for seeds and can retain moisture and/or nutritional components.Examples of characteristics for soils that may be desirable in certainembodiments can be found, for instance, in U.S. Pat. Nos. 3,932,166 and4,707,176. Synthetic plant cultivation media are also well known in theart and may, in certain embodiments, comprise polymers or hydrogels.Examples of such compositions are described, for example, in U.S. Pat.No. 4,241,537.

In certain further aspects, the invention provides plants modified toinclude at least a first trait. Such plants may, in one embodiment, bedeveloped by a plant breeding technique called backcrossing, whereinessentially all of the morphological and physiological characteristicsof a variety are recovered in addition to a genetic locus transferredinto the hybrid via the backcrossing technique. By essentially all ofthe morphological and physiological characteristics, it is meant thatall of the characteristics of a plant are recovered that are otherwisepresent when compared in the same environment, other than an occasionalvariant trait that might arise during backcrossing or directintroduction of a transgene. In one embodiment, such traits may bedetermined, for example, relative to the traits listed in Table 1 asdetermined at the 5% significance level when grown under the sameenvironmental conditions.

Backcrossing methods can be used with the present invention to improveor introduce a trait in a hybrid via modification of its inbredparent(s). The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental corn plants forthat hybrid. The parental corn plant which contributes the locus or locifor the trait is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur.

The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol. In a typical backcrossprotocol, the original parent hybrid of interest (recurrent parent) iscrossed to a second variety (nonrecurrent parent) that carries thegenetic locus of interest to be transferred. The resulting progeny fromthis cross are then crossed again to the recurrent parent and theprocess is repeated until a corn plant is obtained wherein essentiallyall of the morphological and physiological characteristics of therecurrent parent are recovered in the converted plant, in addition tothe transferred locus from the nonrecurrent parent. The backcrossprocess may be accelerated by the use of genetic markers, such as SSR,RFLP, SNP or AFLP markers to identify plants with the greatest geneticcomplement from the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in the original inbred andhybrid progeny therefrom. To accomplish this, a genetic locus of therecurrent parent is modified or substituted with the locus from thenonrecurrent parent, while retaining essentially all of the rest of thegenetic complement, and therefore the morphological and physiologicalconstitution of the original plant. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thecharacteristic has been successfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, waxy starch,herbicide resistance, resistance to bacterial, fungal, or viral disease,insect resistance, male fertility and enhanced nutritional quality.These genes are generally inherited through the nucleus, but may beinherited through the cytoplasm. Some known exceptions to this are genesfor male sterility, some of which are inherited cytoplasmically, butstill act as a single locus trait.

Direct selection may be applied when a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the herbicide resistance characteristic,and only those plants which have the herbicide resistance gene are usedin the subsequent backcross. This process is then repeated for alladditional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofcorn are known to those of skill in the art. For example, methods whichhave been described for the genetic transformation of corn includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. Nos.5,550,318, 5,736,369 and 5,538,880; and PCT Publication WO 95/06128),Agrobacterium-mediated transformation (U.S. Pat. No. 5,591,616 andEuropean Patent Application Publication No. EP0672752), direct DNAuptake transformation of protoplasts and silicon carbide fiber-mediatedtransformation (U.S. Pat. Nos. 5,302,532 and 5,464,765).

Included among various plant transformation techniques are methodspermitting the site-specific modification of a plant genome. Thesemodifications can include, but are not limited to, site-specificmutations, deletions, insertions, and replacements of nucleotides. Thesemodifications can be made anywhere within the genome of a plant, forexample, in genomic elements, including, among others, coding sequences,regulatory elements, and non-coding DNA sequences. Any number of suchmodifications can be made and that number of modifications may be madein any order or combination, for example, simultaneously all together orone after another. Such methods may be used to modify a particular traitconferred by a locus. The techniques for making such modifications bygenome editing are well known in the art and include, for example, useof CRISPR-Cas systems, zinc-finger nucleases (ZFNs), and transcriptionactivator-like effector nucleases (TALENs), among others. Asite-specific nuclease provided herein may be selected from the groupconsisting of a zinc-finger nuclease (ZFN), a meganuclease, anRNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, atransposase, or any combination thereof. See, e.g., Khandagale, K. etal., “Genome editing for targeted improvement in plants,” PlantBiotechnol Rep 10: 327-343 (2016); and Gaj, T. et al., “ZFN, TALEN andCRISPR/Cas-based methods for genome engineering,” Trends Biotechnol.31(7): 397-405 (2013), the contents and disclosures of which areincorporated herein by reference. A recombinase may be a serinerecombinase attached to a DNA recognition motif, a tyrosine recombinaseattached to a DNA recognition motif or other recombinase enzyme known inthe art. A recombinase or transposase may be a DNA transposase orrecombinase attached to a DNA binding domain. A tyrosine recombinaseattached to a DNA recognition motif may be selected from the groupconsisting of a Cre recombinase, a Flp recombinase, and a Tnp1recombinase. According to some embodiments, a Cre recombinase or a Ginrecombinase provided herein is tethered to a zinc-finger DNA bindingdomain. In another embodiment, a serine recombinase attached to a DNArecognition motif provided herein is selected from the group consistingof a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. Inanother embodiment, a DNA transposase attached to a DNA binding domainprovided herein is selected from the group consisting of a TALE-piggyBacand TALE-Mutator. An RNA-guided endonuclease may be selected from thegroup consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7,Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, andhomologs or modified versions thereof, Argonaute (non-limiting examplesof Argonaute proteins include Thermus thermophilus Argonaute (TtAgo),Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryiArgonaute (NgAgo) and homologs or modified versions thereof. Accordingto some embodiments, an RNA-guided endonuclease may be a Cas9 or Cpf1enzyme. For example, the CRISPR/Cas9 system allows targeted cleavage ofgenomic sequences guided by a small noncoding RNA in plants (WO2015026883A1). As another example, Cpf1(Cas12a) acts as anendoribonuclease to process crRNA and an endodeoxyribonuclease to cleavetargeted genomic sequences. The CRISPR/Cpf1 system enables genedeletion, insertion, base editing, and locus tagging in monocot anddicot plants (Alok et al., Frontiers in Plant Science, 31 Mar. 2020).One of ordinary skill in the art of plant breeding would know how tomodify plant genomes using a method including but not limited to thetechniques described herein.

It is understood to those of skill in the art that a transgene or amodified native gene need not be directly transformed into a plant, astechniques for the production of stably transformed corn plants thatpass single loci to progeny by Mendelian inheritance is well known inthe art. Such loci may therefore be passed from parent plant to progenyplants by standard plant breeding techniques that are well known in theart.

A. Male Sterility

Examples of genes conferring male sterility include those disclosed inU.S. Pat. Nos. 3,861,709, 3,710,511, 4,654,465, 5,625,132, and4,727,219, each of the disclosures of which are specificallyincorporated herein by reference in their entirety. Male sterility genescan increase the efficiency with which hybrids are made, in that theyeliminate the need to physically emasculate the corn plant used as afemale in a given cross.

When one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, when cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile Fi hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful when the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns the hybrid corn plant CH011213 comprising a genetic locuscapable of restoring male fertility in an otherwise male-sterile plant.Examples of male-sterility genes and corresponding restorers which couldbe employed with the plants of the invention are well known to those ofskill in the art of plant breeding and are disclosed in, for instance,U.S. Pat. Nos. 5,530,191; 5,689,041; 5,741,684; and 5,684,242, thedisclosures of which are each specifically incorporated herein byreference in their entirety.

B. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. A non-limiting example is a gene conferring resistance toa herbicide that inhibits the growing point or meristem such asimidazolinone or sulfonylurea herbicides. As imidazolinone andsulfonylurea herbicides are acetolactate synthase (ALS)-inhibitingherbicides that prevent the formation of branched chain amino acids,exemplary genes in this category code for ALS and AHAS enzymes asdescribed, for example, by Lee et al., EMBO J., 7:1241, 1988; Gleen etal., Plant Molec. Biology, 18:1185, 1992; and Miki et al., Theor. Appl.Genet., 80:449, 1990. As a non-limiting example, a gene may be employedto confer resistance to the exemplary sulfonylurea herbicidenicosulfuron.

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicusphosphinothricin acetyltransferase (bar) genes) may also be used. See,for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses thenucleotide sequence of a form of EPSPS that can confer glyphosateresistance. Non-limiting examples of EPSPS transformation eventsconferring glyphosate resistance are provided by U.S. Pat. Nos.6,040,497 and 7,632,985. The MON89788 event disclosed in U.S. Pat. No.7,632,985 in particular is beneficial in conferring glyphosate tolerancein combination with an increase in average yield relative to priorevents

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCAccession No. 39256, and the nucleotide sequence of the mutant gene isdisclosed in U.S. Pat. No. 4,769,061 to Comai. A hygromycin Bphosphotransferase gene from E. coli that confers resistance toglyphosate in tobacco callus and plants is described in Penaloza-Vazquezet al., Plant Cell Reports, 14:482, 1995. European Patent ApplicationPublication No. EP0333033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes that confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin acetyltransferase gene isprovided in European Patent Application Publication No. EP0242246 toLeemans et al. DeGreef et al. (Biotechnology, 7:61, 1989) describe theproduction of transgenic plants that express chimeric bar genes codingfor phosphinothricin acetyl transferase activity. Exemplary genesconferring resistance to a phenoxy class herbicide haloxyfop and acyclohexanedione class herbicide sethoxydim are the Acct-S1, Acct-S2 andAcct-S3 genes described by Marshall et al., (Theor. Appl. Genet.,83:435, 1992). As a non-limiting example, a gene may confer resistanceto other exemplary phenoxy class herbicides that include, but are notlimited to, quizalofop-p-ethyl and 2,4-dichlorophenoxyacetic acid(2,4-D).

Genes are also known that confer resistance to herbicides that inhibitphotosynthesis such as, for example, triazine herbicides (psbA and gs+genes) and benzonitrile herbicides (nitrilase gene). As a non-limitingexample, a gene may confer resistance to the exemplary benzonitrileherbicide bromoxynil. Przibila et al. (Plant Cell, 3:169, 1991) describethe transformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (Biochem. J., 285:173, 1992).4-hydroxyphenylpyruvate dioxygenase (HPPD) is a target of theHPPD-inhibiting herbicides, which deplete plant plastoquinone andvitamin E pools. Rippert et al. (Plant Physiol., 134:92, 2004) describesan HPPD-inhibitor resistant tobacco plant that was transformed with ayeast-derived prephenate dehydrogenase (PDH) gene. Protoporphyrinogenoxidase (PPO) is the target of the PPO-inhibitor class of herbicides; aPPO-inhibitor resistant PPO gene was recently identified in Amaranthustuberculatus (Patzoldt et al., PNAS, 103(33):12329, 2006). The herbicidemethyl viologen inhibits CO₂ assimilation. Foyer et al. (Plant Physiol.,109:1047, 1995) describe a plant overexpressing glutathione reductase(GR) that is resistant to methyl viologen treatment.

Siminszky (Phytochemistry Reviews, 5:445, 2006) describes plantcytochrome P450-mediated detoxification of multiple, chemicallyunrelated classes of herbicides. Modified bacterial genes have beensuccessfully demonstrated to confer resistance to atrazine, a herbicidethat binds to the plastoquinone-binding membrane protein QB inphotosystem II to inhibit electron transport. See, for example, studiesby Cheung et al. (PNAS, 85:391, 1988), describing tobacco plantsexpressing the chloroplast psbA gene from an atrazine-resistant biotypeof Amaranthus hybridus fused to the regulatory sequences of a nucleargene, and Wang et al. (Plant Biotech. J., 3:475, 2005), describingtransgenic alfalfa, Arabidopsis, and tobacco plants expressing the atzAgene from Pseudomonas sp. that were able to detoxify atrazine.

Bayley et al. (Theon. Appl. Genet., 83:645, 1992) describe the creationof 2,4-D-resistant transgenic tobacco and cotton plants using the 2,4-Dmonooxygenase gene tfdA from Alcaligenes eutrophus plasmid pJP5. U.S.Patent Application Publication No. 20030135879 describes the isolationof a gene for dicamba monooxygenase (DMO) from Psueodmonas maltophiliathat is involved in the conversion of dicamba to a non-toxic3,6-dichlorosalicylic acid and thus may be used for producing plantstolerant to this herbicide.

Other examples of herbicide resistance have been described, forinstance, in U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114;6,107,549; 5,866,775; 5,804,425; 5,633,435; 5,463,175.

C. Waxy Starch

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1F1, may berequired to determine which plants carry the recessive gene.

D. Disease Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with a cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example, Jones et al.,Science, 266:789, 1994, which describes the cloning of the tomato Cf-9gene for resistance to Cladosporium fulvum; Martin et al., Science,262:1432, 1993, which describes the tomato Pto gene for resistance toPseudomonas syringae pv.; and Mindrinos et al., Cell, 78:1089, 1994,which describes the Arabidopsis RPS2 gene for resistance to Pseudomonassyringae.

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., (Annu. Rev. Phytopathol., 28:451, 1990). Coatprotein-mediated resistance has been conferred upon transformed plantsagainst alfalfa mosaic virus, cucumber mosaic virus, tobacco streakvirus, potato virus X, potato virus Y, tobacco etch virus, tobaccorattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (Nature, 366:469, 1993), who show that transgenicplants expressing recombinant antibody genes are protected from virusattack. Additional means of inducing whole-plant resistance to apathogen include modulation of the systemic acquired resistance (SAR) orpathogenesis related (PR) genes, for example genes homologous to theArabidopsis thaliana NIMI/NPRI/SAI1, and/or by increasing salicylic acidproduction.

Logemann et al., (Biotechnology, 10:305, 1992), for example, disclosetransgenic plants expressing a barley ribosome-inactivating gene have anincreased resistance to fungal disease. Plant defensins may be used toprovide resistance to fungal pathogens (Thomma et al., Planta, 216:193,2002). Other examples of fungal disease resistance are provided in U.S.Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048;5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962.

E. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis (Bt) protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., (Gene,48:109, 1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from the American Type Culture Collection, Manassas, Va.,for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.Another example is a lectin. See, for example, Van Damme et al., (PlantMolec. Biol., 24:825, 1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes. A vitamin-bindingprotein may also be used, such as avidin. See PCT application U.S. Ser.No. 93/06487, the contents of which are hereby incorporated byreference. This application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., (J. Biol. Chem., 262:16793, 1987), which describesthe nucleotide sequence of rice cysteine proteinase inhibitor, Huub etal., (Plant Molec. Biol., 21:985, 1993), which describes the nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I, and Sumitaniet al., (Biosci. Biotech. Biochem., 57:1243, 1993), which describes thenucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).

An insect-specific hormone or pheromone may also be used. See, forexample, Hammock et al., (Nature, 344:458, 1990), which describesbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone, Gade and Goldsworthy (eds.)(Physiological Systems in Insects, Elsevier Academic Press, Burlington,Mass., 2007), which describes allostatins and their potential use inpest control; and Palli et al., (Vitam. Horm., 73:59, 2005), whichdescribes the use of ecdysteroid and ecdysteroid receptor inagriculture. Additionally, the diuretic hormone receptor (DHR) wasidentified in Price et al., (Insect Mol. Biol., 13:469, 2004) as acandidate target of insecticides.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (Seventh Int'l Symposium on Molecular Plant-MicrobeInteractions, Edinburgh, Scotland, Abstract W97, 1994), who describedenzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments.

Nematode resistance has been described, for example, in U.S. Pat. No.6,228,992 and bacterial disease resistance in U.S. Pat. No. 5,516,671.

F. Modified Fatty Acid, Phytate, and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.,(Proc. Natl. Acad. Sci. USA, 89:2624, 1992). Various fatty aciddesaturases have also been described, such as a Saccharomyces cerevisiaeOLE1 gene encoding Δ9 fatty acid desaturase, an enzyme which forms themonounsaturated palmitoleic (16:1) and oleic (18:1) fatty acids frompalmitoyl (16:0) or stearoyl (18:0) CoA (McDonough et al., J. Biol.Chem., 267(9):5931-5936, 1992); a gene encoding a stearoyl-acyl carrierprotein delta-9 desaturase from castor (Fox et al., Proc. Natl. Acad.Sci. USA, 90:2486, 1993); Δ6- and Δ12-desaturases from the cyanobacteriaSynechocystis responsible for the conversion of linoleic acid (18:2) togamma-linolenic acid (18:3 gamma) (Reddy et al., Plant Mol. Biol.,22:293, 1993); a gene from Arabidopsis thaliana that encodes an omega-3desaturase (Arondel et al., Science, 258:1353, 1992); plant Δ9desaturases (PCT Application Publ. No. WO 91/13972) and soybean andBrassica Δ15 desaturases (European Patent Application Publication No.EP0616644).

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (Gene, 127:87, 1993), which discloses the nucleotide sequence ofan Aspergillus niger phytase gene. In corn, this, for example, could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for corn mutants characterized by lowlevels of phytic acid. See Raboy et al., Plant Physiol., 124:355, 1990.

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., (J. Bacteriol., 170:810, 1988), which discloses the nucleotidesequence of Streptococcus mutans fructosyltransferase gene, Steinmetz etal., (Mol. Gen. Genet., 20:220, 1985), which discloses the nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,(Biotechnology, 10:292, 1992), which discloses the production oftransgenic plants that express Bacillus licheniformis α-amylase, Elliotet al., (Plant Molec. Biol., 21:515, 1993), which discloses thenucleotide sequences of tomato invertase genes, Sørgaard et al., (J.Biol. Chem., 268:22480, 1993), which discloses site-directed mutagenesisof barley a-amylase gene, and Fisher et al., (Plant Physiol., 102:1045,1993) which discloses maize endosperm starch branching enzyme II. TheZ10 gene encoding a 10kD zein storage protein from maize may also beused to alter the quantities of 10kD zein in the cells relative to othercomponents (Kirihara et al., Gene, 71:359, 1988).

U.S. Pat. No. 6,930,225 describes maize cellulose synthase genes andmethods of use thereof.

G. Resistance to Abiotic Stress

Abiotic stress includes dehydration or other osmotic stress, salinity,high or low light intensity, high or low temperatures, submergence,exposure to heavy metals, and oxidative stress.Delta-pyrroline-5-carboxylate synthetase (P5CS) from mothbean has beenused to provide protection against general osmotic stress.Mannitol-1-phosphate dehydrogenase (mt1D) from E. coli has been used toprovide protection against drought and salinity. Choline oxidase (codAfrom Arthrobactor globiformis) can protect against cold and salt. E.coli choline dehydrogenase (betA) provides protection against salt.Additional protection from cold can be provided by omega-3-fatty aciddesaturase (fad7) from Arabidopsis thaliana. Trehalose-6-phosphatesynthase and levan sucrase (SacB) from yeast and Bacillus subtilis,respectively, can provide protection against drought (summarized fromAnnex II Genetic Engineering for Abiotic Stress Tolerance in Plants,Consultative Group On International Agricultural Research TechnicalAdvisory Committee). Overexpression of superoxide dismutase can be usedto protect against superoxides, as described in U.S. Pat. No. 5,538,878to Thomas et al.

H. Additional Traits

Additional traits can be introduced into the corn variety of the presentinvention. A non-limiting example of such a trait is a coding sequencewhich decreases RNA and/or protein levels. The decreased RNA and/orprotein levels may be achieved through RNAi methods, such as thosedescribed in U.S. Pat. No. 6,506,559 to Fire et al.

Another trait that may find use with the corn variety of the inventionis a sequence which allows for site-specific recombination. Examples ofsuch sequences include the FRT sequence used with the FLP recombinase;and the LOX sequence used with CRE recombinase. The recombinase genescan be encoded at any location within the genome of the corn plant, andare active in the hemizygous state.

It may also be desirable to make corn plants more tolerant to or moreeasily transformed with Agrobacterium tumefaciens. Expression of p53 andiap, two baculovirus cell-death suppressor genes, inhibited tissuenecrosis and DNA cleavage. Additional targets can include plant-encodedproteins that interact with the Agrobacterium Vir genes; enzymesinvolved in plant cell wall formation; and histones, histoneacetyltransferases and histone deacetylases.

In addition to the modification of oil, fatty acid or phytate contentdescribed above, it may additionally be beneficial to modify the amountsor levels of other compounds. For example, the amount or composition ofantioxidants can be altered. See, for example, U.S. Pat. Nos. 6,787,618and 7,154,029 and International Patent Application Publication No. WO00/68393, which disclose the manipulation of antioxidant levels, andInternational Patent Application. Publication No. WO 03/082899, whichdiscloses the manipulation of an antioxidant biosynthetic pathway.

Additionally, seed amino acid content may be manipulated. U.S. Pat. No.5,850,016 and International Patent Application Publication No. WO99/40209 disclose the alteration of the amino acid compositions ofseeds. U.S. Pat. Nos. 6,080,913 and 6,127,600 disclose methods ofincreasing accumulation of essential amino acids in seeds.

U.S. Pat. No. 5,559,223 describes synthetic storage proteins in whichthe levels of essential amino acids can be manipulated. InternationalPatent Application Publication No. WO 99/29882 discloses methods foraltering amino acid content of proteins. International PatentApplication Publication No. WO 98/20133 describes proteins with enhancedlevels of essential amino acids. International Patent ApplicationPublication No. WO 98/56935 and U.S. Pat. Nos. 6,346,403, 6,441,274 and6,664,445 disclose plant amino acid biosynthetic enzymes. InternationalPatent Application Publication No. WO 98/45458 describes synthetic seedproteins having a higher percentage of essential amino acids thanwild-type.

U.S. Pat. No. 5,633,436 discloses plants comprising a higher content ofsulfur-containing amino acids; U.S. Pat. No. 5,885,801 discloses plantscomprising a high threonine content; U.S. Pat. No. 5,885,802 disclosesplants comprising a high methionine content; U.S. Pat. No. 5,912,414discloses plants comprising a high methionine content; U.S. Pat. No.5,990,389 discloses plants comprising a high lysine content; U.S. Pat.No. 6,459,019 discloses plants comprising an increased lysine andthreonine content; International Patent Application Publication No. WO98/42831 discloses plants comprising a high lysine content;International Patent Application Publication No. WO 96/01905 disclosesplants comprising a high threonine content; and International PatentApplication Publication No. WO 95/15392 discloses plants comprising ahigh lysine content.

I. Origin and Breeding History of an Exemplary Introduced Trait

Provided by the invention are a hybrid plant in which one or more of theparents comprise an introduced trait. Such a plant may be defined ascomprising a single locus conversion. Exemplary procedures for thepreparation of such single locus conversions are disclosed in U.S. Pat.No. 7,205,460, the entire disclosure of which is specificallyincorporated herein by reference.

An example of a single locus conversion is 85DGD1. 85DGD1 MLms is aconversion of 85DGD1 to cytoplasmic male sterility. 85DGD1 MLms wasderived using backcross methods. 85DGD1 (a proprietary inbred ofMonsanto Company) was used as the recurrent parent and MLms, a germplasmsource carrying ML cytoplasmic sterility, was used as the nonrecurrentparent. The breeding history of the converted inbred 85DGD1 MLms can besummarized as follows:

Hawaii Nurseries Planting Made up S-O: Female row 585 male row 500 DateApr. 2, 1992 Hawaii Nurseries Planting S-O was grown and plants werebackcrossed Date Jul. 15, 1992 times 85DGD1 (rows 444 {acute over ( )}443) Hawaii Nurseries Planting Bulked seed of the BC₁ was grown and DateNov. 18, 1992 backcrossed times 85DGD1 (rows V3-27 {acute over ( )}V3-26) Hawaii Nurseries Planting Bulked seed of the BC₂ was grown andDate Apr. 2, 1993 backcrossed times 85DGD1 (rows 37 {acute over ( )} 36)Hawaii Nurseries Planting Bulked seed of the BC₃ was grown and Date Jul.14, 1993 backcrossed times 85DGD1 (rows 99 {acute over ( )} 98) HawaiiNurseries Planting Bulked seed of BC₄ was grown and Date Oct. 28, 1993backcrossed times 85DGD1 (rows KS-63 {acute over ( )} KS-62) Summer 1994A single ear of the BC₅ was grown and backcrossed times 85DGD1 (MC94-822{acute over ( )} MC94-822-7) Winter 1994 Bulked seed of the BC₆ wasgrown and backcrossed times 85DGD1 (3Q-1 {acute over ( )} 3Q-2) Summer1995 Seed of the BC₇ was bulked and named 85DGD1 MLms.

As described, techniques for the production of corn plants with addedtraits are well known in the art. A non-limiting example of such aprocedure one of skill in the art could use for preparation of a hybridcorn plant CH011213 comprising an added trait is as follows:

-   -   (a) crossing a parent of hybrid corn plant CH011213 such as        CV621679 and/or CV512858 to a second (nonrecurrent) corn plant        comprising a locus to be converted in the parent;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to the parent line of corn        plant CH011213;    -   (d) repeating steps (b) and (c) until a parent line of variety        CH011213 is obtained comprising the locus; and    -   (e) crossing the converted parent with the second parent to        produce hybrid variety CH011213 comprising a trait.

Following these steps, essentially any locus may be introduced intohybrid corn variety CH011213. For example, molecular techniques allowintroduction of any given locus, without the need for phenotypicscreening of progeny during the backcrossing steps.

PCR and Southern hybridization are two examples of molecular techniquesthat may be used for confirmation of the presence of a given locus andthus conversion of that locus. The techniques are carried out asfollows: Seeds of progeny plants are grown and DNA isolated from leaftissue. Approximately one gram of leaf tissue is lyophilized overnightin 15 ml polypropylene tubes. Freeze-dried tissue is ground to a powderin the tube using a glass rod. Powdered tissue is mixed thoroughly with3 ml extraction buffer (7.0M urea, 0.35M NaCl, 0.05M Tris-HCI pH 8.0,0.01M EDTA, 1% sarcosine). Tissue/buffer homogenate is extracted with 3ml phenol/chloroform. The aqueous phase is separated by centrifugation,and precipitated twice using 1/10 volume of 4.4M ammonium acetate pH5.2, and an equal volume of isopropanol. The precipitate is washed with75% ethanol and resuspended in 100-500 μl TE (0.01M Tris-HCI, 0.001MEDTA, pH 8.0). The DNA may then be screened as desired for presence ofthe locus.

For PCR, 200-1000 ng genomic DNA from the progeny plant being screenedis added to a reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mMMgCl₂ , 50mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP, dGTP, dTTP,20% glycerol, 2.5 units Taq DNA polymerase and 0.5 μM each of forwardand reverse DNA primers that span a segment of the locus beingconverted. The reaction is run in a thermal cycling machine 3 minutes at94 C, 39 repeats of the cycle 1 minute at 94 C, 1 minute at 50 C, 30seconds at 72 C, followed by 5 minutes at 72 C. Twenty μl of eachreaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mMTris-borate, 2 mM EDTA) at 50V for two to four hours. The amplifiedfragment is detected using an agarose gel. Detection of an amplifiedfragment corresponding to the segment of the locus spanned by theprimers indicates the presence of the locus.

For Southern analysis, plant DNA is restricted, separated in an agarosegel and transferred to a Nylon filter in 10× SCP (20 SCP: 2M NaCl, 0.6Mdisodium phosphate, 0.02M disodium EDTA) according to standard methods(Southern, J. Mol. Biol., 98:503, 1975). Locus DNA or RNA sequences arelabeled, for example, radioactively with ³²P by random priming (Feinberg& Vogelstein, Anal. Biochem., 132(1):6, 1983). Filters are prehybridizedin 6× SCP, 10% dextran sulfate, 2% sarcosine, and 500 μg/ml denaturedsalmon sperm DNA. The labeled probe is denatured, hybridized to thefilter and washed in 2× SCP, 1% SDS at 65° C. for 30 minutes andvisualized by autoradiography using Kodak XAR5 film. Presence of thelocus is indicated by detection of restriction fragments of theappropriate size.

Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated CH011213. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In one embodiment, thetissue culture comprises embryos, protoplasts, meristematic cells,pollen, leaves or anthers derived from immature tissues of these plantparts. Means for preparing and maintaining plant tissue cultures arewell known in the art (U.S. Pat. Nos. 5,538,880 and 5,550,318, eachincorporated herein by reference in their entirety). By way of example,a tissue culture comprising organs such as tassels or anthers has beenused to produce regenerated plants (U.S. Pat. Nos. 5,445,961 and5,322,789; the disclosures of which are incorporated herein byreference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they can be selected at a stage when the microspores areuninucleate, that is, include only 1, rather than 2 or 3 nuclei. Methodsto determine the correct stage are well known to those skilled in theart and include mitramycin fluorescent staining, trypan blue, andacetocarmine squashing. The mid-uninucleate microspore stage has beenfound to be the developmental stage most responsive to the subsequentmethods disclosed to ultimately produce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4° C. to 25° C. may be preferred, and a range of 8° C. to 14°C. may be particularly preferred. Although other temperatures yieldembryoids and regenerated plants, cold temperatures produce optimumresponse rates compared to pretreatment at temperatures outside thepreferred range. Response rate is measured as either the number ofembryoids or the number of regenerated plants per number of microsporesinitiated in culture. Exemplary methods of microspore culture aredisclosed in, for example, U.S. Pat. Nos. 5,322,789 and 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally beneficial to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are generallypretreated at a cold temperature for a predefined time, often at 10° C.for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In one embodiment, 3 ml of 0.3M mannitol combined with 50 mg/l ofascorbic acid, silver nitrate, and colchicine is used for incubation ofanthers at 10° C. for between 10 and 14 days. Another embodiment is tosubstitute sorbitol for mannitol. The colchicine produces chromosomedoubling at this early stage. The chromosome doubling agent is generallyonly present at the preculture stage.

It is believed that the mannitol or other similar carbon structures orenvironmental stress induce starvation and function to force microsporesto focus their energies on entering developmental stages. The cells areunable to use, for example, mannitol as a carbon source at this stage.It is believed that these treatments confuse the cells causing them todevelop as embryoids and plants from microspores. Dramatic increases indevelopment from these haploid cells, as high as 25 embryoids in 10⁴microspores, have resulted from using these methods.

To isolate microspores, an isolation media is generally used. Anisolation media is used to separate microspores from the anther wallswhile maintaining their viability and embryogenic potential. Anillustrative embodiment of an isolation media includes a 6% sucrose ormaltose solution combined with an antioxidant such as 50 mg/l ofascorbic acid, 0.1 mg/l biotin, and 400 mg/l of proline, combined with10 mg/l of nicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, thebiotin and proline are omitted.

An isolation media preferably has a higher antioxidant level when it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. An illustrativeembodiment of a solid support is a TRANSWELL® culture dish. Anotherembodiment of a solid support for development of the microspores is abilayer plate wherein liquid media is on top of a solid base. Otherembodiments include a mesh or a millipore filter. Preferably, a solidsupport is a nylon mesh in the shape of a raft. A raft is defined as anapproximately circular support material which is capable of floatingslightly above the bottom of a tissue culture vessel, for example, apetri dish, of about a 60 or 100 mm size, although any other laboratorytissue culture vessel will suffice. In an illustrative embodiment, araft is about 55 mm in diameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent).

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application Publication No.EP0160390, PCT Application WO 95/06128, and U.S. Pat. No. 5,736,369.

Processes of Crossing Corn Plants and the Corn Plants Produced by SuchCrosses

The present invention provides processes of preparing novel corn plantsand corn plants produced by such processes. In accordance with such aprocess, a first parent corn plant may be crossed with a second parentcorn plant wherein the first and second corn plants are the parent linesof hybrid corn plant variety CH011213, or wherein at least one of theplants is of hybrid corn plant variety CH011213.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when the wind blowspollen from the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand. In one embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, when desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. When the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant CH011213 is employedas the male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine. Alternatively, when the female parent corn plantcomprises a cytoplasmic or nuclear gene conferring male sterility,detasseling may not be required. Additionally, a chemical gametocide maybe used to sterilize the male flowers of the female plant. In this case,the parent plants used as the male may either not be treated with thechemical agent or may comprise a genetic factor which causes resistanceto the emasculating effects of the chemical agent. Gametocides affectprocesses or cells involved in the development, maturation or release ofpollen. Plants treated with such gametocides are rendered male sterile,but typically remain female fertile. The use of chemical gametocides isdescribed, for example, in U.S. Pat. No. 4,936,904, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.Furthermore, the use of Roundup herbicide in combination with glyphosatetolerant corn plants to produce male sterile corn plants is disclosed inPCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the plant being used as the femalein the hybridization. Of course, during this hybridization procedure,the parental varieties are grown such that they are isolated from othercorn fields to minimize or prevent any accidental contamination ofpollen from foreign sources. These isolation techniques are well withinthe skill of those skilled in this art.

Both parental plants of corn may be allowed to continue to grow untilmaturity or the male rows may be destroyed after flowering is complete.Only the ears from the female parental plants are harvested to obtainseeds of a novel F₁ hybrid. The novel F₁ hybrid seed produced can thenbe planted in a subsequent growing season in commercial fields or,alternatively, advanced in breeding protocols for purposes of developingnovel inbred lines.

Alternatively, in another embodiment of the invention, one or both firstand second parent corn plants can be from variety CH011213. Thus, anycorn plant produced using corn plant CH011213 forms a part of theinvention. As used herein, crossing can mean selfing, backcrossing,crossing to another or the same variety, crossing to populations, andthe like. All corn plants produced using the corn variety CH011213 as aparent are, therefore, within the scope of this invention.

One use of the instant corn variety is in the production of hybrid seed.Any time the corn plant CH011213 is crossed with another, different,corn plant, a corn hybrid plant is produced. As such, hybrid corn plantcan be produced by crossing CH011213 with any second corn plant.Essentially any other corn plant can be used to produce a corn planthaving corn plant CH011213 as one parent. All that is required is thatthe second plant be fertile, which corn plants naturally are, and thatthe plant is not corn variety CH011213.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

The development of new inbred varieties using one or more startingvarieties is well known in the art. In accordance with the invention,novel varieties may be created by crossing a corn variety, followed bymultiple generations of breeding according to such well known methods.New varieties may be created by crossing a corn variety with any secondplant. In selecting such a second plant to cross for the purpose ofdeveloping novel inbred lines, it may be desired to choose those plantswhich either themselves exhibit one or more desirable characteristics orwhich exhibit the desirable characteristic(s) when in hybridcombination. Examples of potentially desirable characteristics includegreater yield, better stalks, better roots, resistance to insecticides,herbicides, pests, and disease, tolerance to heat and drought, reducedtime to crop maturity, better agronomic quality, higher nutritionalvalue, and uniformity in germination times, stand establishment, growthrate, maturity, and fruit size.

Once initial crosses have been made with a corn variety, inbreedingtakes place to produce new inbred varieties. Inbreeding requiresmanipulation by human breeders. Even in the extremely unlikely eventinbreeding rather than crossbreeding occurred in natural corn,achievement of complete inbreeding cannot be expected in nature due towell-known deleterious effects of homozygosity and the large number ofgenerations the plant would have to breed in isolation. The reason forthe breeder to create inbred plants is to have a known reservoir ofgenes whose gametic transmission is predictable.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desirable characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection. After at least five generations, the inbred plant isconsidered genetically pure.

Marker assisted selection (MAS) can be used to reduce the number ofbreeding cycles and improve selection accuracy. For example, seeOpenshaw et al., “Marker-assisted Selection in Backcross Breeding,” in:Proceedings Symposium of the Analysis of Molecular Data, August 1994,Crop Science Society of America. Genome-wide selection (GWS)/genomicselection (GS) can also be used as an alternative to, or in combinationto, marker assisted selection and phenotype selection. GS utilizesquantitative models over a large number of markers distributed acrossthe genome to predict the genomic estimated breeding values (GEBVs) ofindividual plants that has been genotyped but not phenotyped. GS canimprove complex traits or combination of multiple traits without theneed to identify markers associated with the traits. GS can replacephenotyping for a few selection cycles, thus reducing the cost and thetime required for variety development (Crossa et al., Trends in PlantScience, November 2017, Vol. 22, No.11).

Uniform lines of new varieties may also be developed by way ofdoubled-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid induction systems have been developedfor various plants to produce haploid tissues, plants and seeds. Thehaploid induction system can produce haploid plants from any genotype bycrossing with an inducer line. Inducer lines and methods for obtaininghaploid plants are known in the art.

Haploid embryos may be produced, for example, from microspores, pollen,anther cultures, or ovary cultures. The haploid embryos may then bedoubled autonomously, or by chemical treatments (e.g. colchicinetreatment). Alternatively, haploid embryos may be grown into haploidplants and treated to induce chromosome doubling. In either case,fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the invention and progeny thereof to achieve a homozygous line.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their inbredparents. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields, better stalks,better roots, better uniformity and better insect and diseaseresistance. In the development of hybrids only the F₁ hybrid plants aretypically sought. An F₁ single cross hybrid is produced when two inbredplants are crossed. A double cross hybrid is produced from four inbredplants crossed in pairs (A x B and C x D) and then the two F₁ hybridsare crossed again (A×B)×(C×D).

Thousands of corn varieties are known to those of skill in the art, anyone of which could be crossed with corn plant CH011213 to produce ahybrid plant. Estimates place the number of different corn accessions ingene banks around the world at around 50,000. The Maize GeneticsCooperation Stock Center, which is supported by the U.S. Department ofAgriculture, has a total collection of over 80,000 individuallypedigreed samples (available on the World Wide Web atmaizecoop.cropsci.uiuc.edu/).

When the corn plant CH011213 is crossed with another plant to yieldprogeny, it can serve as either the maternal or paternal plant. For manycrosses, the outcome is the same regardless of the assigned sex of theparental plants. However, due to increased seed yield and productioncharacteristics, it may be desired to use one parental plant as thematernal plant. Some plants produce tighter ear husks leading to moreloss, for example due to rot. There can be delays in silk formationwhich deleteriously affect timing of the reproductive cycle for a pairof parental inbreds. Seed coat characteristics can be preferable in oneplant. Pollen can be shed better by one plant. Other variables can alsoaffect preferred sexual assignment of a particular cross.

The development of a hybrid corn variety involves three steps: (1)selecting plants from various germplasm pools; (2) selfing the selectedplants for several generations to produce a series of inbred plants,which although different from each other, each breed true and are highlyuniform; and (3) crossing the selected inbred plants with unrelatedinbred plants to produce F₁ hybrid progeny. During this inbreedingprocess in corn, the vigor of the plants decreases; however, vigor isrestored when two unrelated inbred plants are crossed to produce F₁hybrid progeny. An important consequence of the genetic homozygosity andhomogeneity of an inbred plant is that the F₁ hybrid progeny of any twoinbred varieties are genetically and phenotypically uniform. Plantbreeders choose these hybrid populations that display phenotypicuniformity. Once the inbred plants that produce superior hybrid progenyhave been identified, the uniform traits of their hybrid progeny can bereproduced indefinitely as long as the homogeneity of the inbred parentsis maintained.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by Monsanto Company to perform the final evaluation ofthe commercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further development of theexperimental hybrids. After FACT testing is complete, determinations maybe made whether commercial development should proceed for a givenhybrid.

The present invention provides a genetic complement of the hybrid cornplant variety designated CH011213. As used herein, the phrase “geneticcomplement” means an aggregate of nucleotide sequences, the expressionof which defines the phenotype of a corn plant or a cell or tissue ofthat plant. By way of example, a corn plant is genotyped to determine arepresentative sample of the inherited markers it possesses. Markers arealleles at a single locus. They are preferably inherited in codominantfashion so that the presence of both alleles at a diploid locus isreadily detectable, and they are free of environmental variation, i.e.,their heritability is 1. This genotyping is preferably performed on atleast one generation of the descendant plant for which the numericalvalue of the quantitative trait or traits of interest are alsodetermined. The array of single locus genotypes is expressed as aprofile of marker alleles, two at each locus. The marker alleliccomposition of each locus can be either homozygous or heterozygous.Homozygosity is a condition in which both alleles at a locus arecharacterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

The references cited herein, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

What is claimed is:
 1. A seed of hybrid corn variety CH011213, producedby crossing a first plant of variety CV621679 with a second plant ofvariety CV512858, wherein representative seeds of said varietiesCV621679 and CV512858 are deposited under ATCC Accession No. PTA-125246and NCMA Accession No. 202106028, respectively.
 2. A plant of the hybridcorn variety CH011213 grown from said seed of claim
 1. 3. A plant partof the plant of claim 2, wherein said plant part comprises a cell ofsaid hybrid corn variety CH011213.
 4. A composition comprising the seedof claim 1 comprised in plant seed growth media.
 5. The composition ofclaim 4, wherein said growth media is soil or a synthetic cultivationmedium.
 6. The seed of claim 1, further comprising a transgene, whereinsaid transgene is introduced by backcrossing or genetic transformationinto said variety CV621679, said variety CV512858, or both varietiesCV621679 and CV512858.
 7. The seed of claim 6, wherein the transgeneconfers a trait selected from the group consisting of male sterility,herbicide tolerance, insect resistance, disease resistance, waxy starch,modified fatty acid metabolism, modified phytic acid metabolism,modified carbohydrate metabolism and modified protein metabolism.
 8. Amethod of producing the seed of claim 1, the method comprising crossinga plant of variety CV621679 with a plant of variety CV512858.
 9. A seedof hybrid corn variety CH011213 further comprising a single locusconversion, wherein a plant grown from said seed comprises a traitconferred by said single locus conversion and otherwise comprises all ofthe morphological and physiological characteristics of hybrid cornvariety CH011213, and wherein said seed is produced by crossing a firstplant selected from the group consisting of variety CV621679 and selfedprogeny thereof with a second plant selected from a second groupconsisting of variety CV512858 and selfed progeny thereof, wherein saidfirst plant, said second plant, or both further comprise said singlelocus conversion, and wherein representative seeds of said varietiesCV621679 and CV512858 are deposited under ATCC Accession No. PTA-125246and NCMA Accession No. 202106028, respectively.
 10. The seed of claim 9,wherein said single locus conversion confers a trait selected from thegroup consisting of male sterility, herbicide tolerance, insectresistance, disease resistance, waxy starch, modified fatty acidmetabolism, modified phytic acid metabolism, modified carbohydratemetabolism and modified protein metabolism.
 11. A plant grown from theseed of claim 9, wherein said plant comprises said trait and otherwisecomprises all of the morphological and physiological characteristics ofhybrid corn variety CH011213.
 12. A method of introducing a heritabletrait into hybrid corn variety CH011213, the method comprising the stepsof: (a) introducing at least said heritable trait into a first inbredcorn variety CV621679, a second inbred corn variety CV512858, or bothinbred corn varieties CV621679 and CV512858 to produce plants of saidinbred corn varieties that heritably carry said heritable trait, whereinsaid heritable trait is introduced into said inbred corn varieties bybackcrossing, wherein said backcrossing is sufficient to produce aninbred corn variety further comprising said heritable trait, and whereinrepresentative seeds of said inbred corn varieties CV621679 and CV512858are deposited under ATCC Accession No. PTA-125246 and NCMA Accession No.202106028, respectively; and (b) producing a plant of hybrid cornvariety CH011213 further comprising said heritable trait by crossing aplant of said first or said second inbred corn variety that heritablycarries said heritable trait with a plant of a different inbred cornvariety selected from a group consisting of inbred corn varietiesCV621679 and CV512858, or crossing a plant of said first inbred cornvariety and a plant of said second inbred corn variety that bothheritably carry said heritable trait.
 13. The method of claim 12 whereinsaid heritable trait is selected from the group consisting of malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism.
 14. The method of claim 12 further comprising repeating step(a) at least once to introduce at least a second heritable trait intohybrid corn variety CH011213, wherein the second heritable trait isselected from the group consisting of male sterility, herbicidetolerance, insect resistance, disease resistance, waxy starch, modifiedfatty acid metabolism, modified phytic acid metabolism, modifiedcarbohydrate metabolism and modified protein metabolism.
 15. A plantproduced by the method of claim 12, wherein said plant comprises saidheritable trait and otherwise comprises all of the morphological andphysiological characteristics of corn variety CH011213 when grown underthe same environmental conditions.
 16. A method of producing a progenycorn plant derived from hybrid corn variety CH011213, wherein the methodcomprises applying plant breeding techniques to the plant of claim 2 toproduce said progeny corn plant derived from hybrid corn varietyCH011213.
 17. The method of claim 16, wherein said plant breedingtechniques comprise backcrossing, marker assisted breeding, pedigreebreeding, selfing, outcrossing, haploid production, doubled haploidproduction, or transformation.
 18. The method of claim 16, furthercomprising the steps of: (a) crossing said progeny corn plant derivedfrom hybrid corn variety CH011213 with itself or a second plant toproduce a seed of a progeny plant of a subsequent generation; (b)growing the progeny plant of the subsequent generation from said seed ofthe progeny plant of the subsequent generation; and (c) repeating steps(a) and (b) for at least an additional 3-10 generations to produce aprogeny corn plant further derived from the hybrid corn varietyCH011213.
 19. A method of producing a commodity plant product, themethod comprising obtaining the plant of claim 2 or a part thereof andproducing said commodity plant product therefrom wherein said plant partcomprises a cell of hybrid corn variety CH011213.
 20. The method ofclaim 19, wherein said commodity plant product is grain, starch, seedoil, corn syrup, or protein.